PI(4,5)P 2 role in Transverse-tubule membrane formation and muscle function.

Transverse (T)-tubules - vast, tubulated domains of the muscle plasma membrane - are critical to maintain healthy skeletal and heart contractions. How the intricate T-tubule membranes are formed is not well understood, with challenges to systematically interrogate in muscle. We established the use of intact Drosophila larval body wall muscles as an ideal system to discover mechanisms that sculpt and maintain the T-tubule membrane network. A muscle-targeted genetic screen identified specific phosphoinositide lipid regulators necessary for T-tubule organization and muscle function. We show that a PI4KIIIα - Skittles/PIP5K pathway is needed for T-tubule localized PI(4)P to PI(4,5)P 2 synthesis, T-tubule organization, calcium regulation, and muscle and heart rate functions. Muscles deficient for PI4KIIIα or Amphiphysin , the homolog of human BIN1 , similarly exhibited specific loss of transversal T-tubule membranes and dyad junctions, yet retained longitudinal membranes and the associated dyads. Our results highlight the power of live muscle studies, uncovering distinct mechanisms and functions for sub-compartments of the T-tubule network relevant to human myopathy. Summary: T-tubules - vast, tubulated domains of the muscle plasma membrane - are critical to maintain skeletal and heart contractions. Fujita et al . establish genetic screens and assays in intact Drosophila muscles that uncover PI(4,5)P 2 regulation critical for T-tubule maintenance and function. Key Findings: PI4KIIIα is required for muscle T-tubule formation and larval mobility. A PI4KIIIα-Sktl pathway promotes PI(4)P and PI(4,5)P 2 function at T-tubules. PI4KIIIα is necessary for calcium dynamics and transversal but not longitudinal dyads. Disruption of PI(4,5)P 2 function in fly heart leads to fragmented T-tubules and abnormal heart rate.

MS-275, a class 1 histone deacetylase inhibitor augments glucagon-like peptide-1 receptor agonism to improve glycemic control and reduce obesity in diet-induced obese mice.

Given its glycemic efficacy and ability to reduce the body weight, glucagon-like peptide 1 receptor (GLP-1R) agonism has emerged as a preferred treatment for diabetes associated with obesity. We here report that a small-molecule Class 1 histone deacetylase (HDAC) inhibitor Entinostat (MS-275) enhances GLP-1R agonism to potentiate glucose-stimulated insulin secretion and decrease body weight in diet-induced obese (DIO) mice. MS-275 is not an agonist or allosteric activator of GLP-1R but enhances the sustained receptor-mediated signaling through the modulation of the expression of proteins involved in the signaling pathway. MS-275 and liraglutide combined therapy improved fasting glycemia upon short-term treatment and a chronic administration causes a reduction of obesity in DIO mice. Overall, our results emphasize the therapeutic potential of MS-275 as an adjunct to GLP-1R therapy in the treatment of diabetes and obesity.

Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment.

OBJECTIVE: Upon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells. METHODS: We studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein-protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes. RESULTS: Our data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment. CONCLUSIONS: The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex.

Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells.

Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation.

Glucagon-like peptide-1 receptor-mediated endosomal cAMP generation promotes glucose-stimulated insulin secretion in pancreatic ß-cells.

Glucagon-like peptide-1 receptor (GLP-1R) plays a major role in promoting glucose-stimulated insulin secretion in pancreatic ß-cells. In the present study, we synthesized a novel functional analog of GLP-1 conjugated to tetramethyl rhodamine to monitor the internalization of the receptor. Our data show that after being internalized the receptor is sorted to lysosomes. In endosomes, receptor-ligand complex is found to be colocalized with adenylate cyclase. Pharmacological inhibition of endocytosis attenuates GLP-1R-mediated cAMP generation and consequent downstream protein kinase A substrate phosphorylation and glucose-stimulated insulin secretion. Our study underlines a paradigm shift in GLP-1R signaling and trafficking. The receptor ligand complex triggers cAMP generation both in plasma membrane and in endosomes, which has implications for receptor-mediated regulation of insulin secretion.